Fatty acid ?-oxidation is the major energy-producing process in the liver, heart, and muscle. It is carried out by a series of four reactions that successively cleave acetyl-CoA from fatty acyl-CoA. The rate of this process can be altered by diet (fed/fasting), physiological status (pregnancy), or diseases (diabetes). The first of the four reactions in this process is initiated by a family of flavoproteins, acyl-CoA dehydrogenases (ADs). Electron transfer from ADs to the mitochondrial OXPHOS chain is catalyzed by electron transfer flavoprotein (ETF) and the membrane-bound ETF-ubiquinoneoxidoreductase (ETF-QO). There are at least seven soluble ADs for catalyzing short chain acyl-CoAs, and two membrane-bound ADs specific for long chain fatty acyl-CoAs, very long chain AD (VLCAD) and ACAD9. The three remaining reactions of ?- oxidation for long chain fatty acids are carried out by the trifunctional protein (TFP), a membrane-bound multienzyme complex. Inborn errors of fatty acid oxidation have emerged as an increasing health problem and now represent the most common group of disorders identified through expanded newborn screening, affecting 2-3/1,000 babies born nationwide. These disorders present sudden infant death syndrome, cause cardiomyopathy, and are the most common cause of skeletal myopathy in older children and young adults. Recently ACAD9 has been shown to be essential for the assembly of Complex I, the largest and most complicated enzyme (~980 kDa with 45 subunits) among the five OXPHOS complexes. Very little is known concerning the mechanism of the assembly process of this important enzyme. Disorders of the mitochondrial OXPHOS system are the most common of inborn metabolic diseases, resulting in a wide variety of clinical phenotypes ranging from exercise intolerance to failure to thrive. We have determined the crystal structures of all but one of the soluble ADs, as well as one membrane-bound AD (VLCAD), ETF, and ETF-QO. The proposed investigations are focused on three membrane-bound enzymes, VLCAD, ACAD9, and TFP, and interactions of ETF with its electron transfer partners, including ADs, dimethylglycine dehydrogenase (DD), and ETF-QO. DD functions in choline metabolism and is not a member of the AD family, but donates electrons to ETF. Specific Aims are: 1) Structural studies of human TFP by X-ray crystallography to understand how its three distinct active sites communicate with each other; 2) Studies of VLCAD, including a) studies of clinical mutations, and b) to determine the orientation of VLCAD on the mitochondrial membrane and interactions with TFP and ETF by EPR spectroscopy; 3) studies of ACAD9 to determine the biochemical/structural basis for its unique role in mitochondrial Complex I assembly; and 4) to investigate the domain movement of ETF and its interactions with three representative electron donors (medium chain acyl-CoA dehydrogenase, VLCAD, and DD) and with its electron acceptor, ETF-QO. PUBLIC HEALTH RELEVANCE: All cells in the human body need energy to maintain their structural integrity and to perform their vital functions. The major proportion of this energy comes from the oxidation of fatty acids in heart, liver, and muscle cells and an imbalance in this process results in disease states, such as obesity and diabetes. A better understanding of how these enzymes function will provide insight into diagnosis and the design and development of novel inhibitors or agonists for the treatment of metabolic disorders.